BACKGROUND: Additional urinary biomarkers for diabetic nephropathy (DN) are needed, providing early and reliable diagnosis and new insights into its mechanisms. Rigorous selection criteria and homogeneous study population may improve reproducibility of the proteomic approach. METHODS: Long-term type 1 diabetes patients without metabolic comorbidities were included, 11 with sustained microalbuminuria (MA) and 14 without MA (nMA). Morning urine proteins were precipitated and resolved by 2D electrophoresis. Principal component analysis (PCA) and Projection to latent structures discriminatory analysis (PLS-DA) were adopted to assess general data validity, to pick protein fractions for identification with mass spectrometry (MS), and to test predictive value of the resulting model. RESULTS: Proteins (n = 113) detected in more than 90% patients were considered representative. Unsupervised PCA showed excellent natural data clustering without outliers. Protein spots reaching Variable Importance in Projection score above 1 in PLS (n = 42) were subjected to MS, yielding 33 positive identifications. The PLS model rebuilt with these proteins achieved accurate classification of all patients (R2X = 0.553, R2Y = 0.953, Q2 = 0.947). Thus, multiple earlier recognized biomarkers of DN were confirmed and several putative new biomarkers suggested. Among them, the highest significance was met in kininogen-1. Its activation products detected in nMA patients exceeded by an order of magnitude the amount found in MA patients. CONCLUSIONS: Reducing metabolic complexity of the diseased and control groups by meticulous patients' selection allows to focus the biomarker search in DN. Suggested new biomarkers, particularly kininogen fragments, exhibit the highest degree of correlation with MA and substantiate validation in larger and more varied cohorts.

Department of Internal Medicine 1 Charles University School of Medicine in Pilsen alej Svobody 80 Pilsen 304 60 Czech Republic

Department of Internal Medicine Teaching Hospital Motol 5 Uvalu 84 Prague 5 150 06 Czech Republic

Proteomic Laboratory Charles University School of Medicine in Pilsen alej Svobody 1655 76 Pilsen 323 00 Czech Republic

Zobrazit více v PubMed

Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature. 2001;414:782–787. doi: 10.1038/414782a. PubMed DOI

Caramori ML, Fioretto P, Mauer M. The need for early predictors of diabetic nephropathy risk: is albumin excretion rate sufficient? Diabetes. 2000;49:1399–1408. doi: 10.2337/diabetes.49.9.1399. PubMed DOI

Molitch ME, Steffes M, Sun W, et al. Development and progression of renal insufficiency with and without albuminuria in adults with type 1 diabetes in the diabetes control and complications trial and the epidemiology of diabetes interventions and complications study. Diabetes Care. 2010;33:1536–1543. doi: 10.2337/dc09-1098. PubMed DOI PMC

Ruggenenti P, Remuzzi G. Time to abandon microalbuminuria? Kidney Int. 2006;70:1214–1222. doi: 10.1038/sj.ki.5001729. PubMed DOI

Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome: a broken mirror of renal diseases. J Am Soc Nephrol. 2007;18:2233–2239. doi: 10.1681/ASN.2006121399. PubMed DOI

Pisitkun T, Johnstone R, Knepper MA. Discovery of urinary biomarkers. Mol Cell Proteomics. 2006;5:1760–1771. doi: 10.1074/mcp.R600004-MCP200. PubMed DOI

Ben Ameur R, Molina L, Bolvin C, et al. Proteomic approaches for discovering biomarkers of diabetic nephropathy. Nephrol Dial Transplant. 2010;25:2866–2875. doi: 10.1093/ndt/gfq258. PubMed DOI

Mares J, Thongboonkerd V, Tuma Z, et al. Specific adsorption of some complement activation proteins to polysulfone dialysis membranes during hemodialysis. Kidney Int. 2009;76:404–413. doi: 10.1038/ki.2009.138. PubMed DOI

Norden B, Broberg P, Lindberg C, Plymoth A. Analysis and understanding of high-dimensionality data by means of multivariate data analysis. Chem Biodivers. 2005;2:1487–1494. doi: 10.1002/cbdv.200590120. PubMed DOI

Kitano H, Oda K, Kimura T, et al. Metabolic syndrome and robustness tradeoffs. Diabetes. 2004;53(Suppl 3):S6–S15. doi: 10.2337/diabetes.53.suppl_3.S6. PubMed DOI

Janech MG, Raymond JR, Arthur JM. Proteomics in renal research. Am J Physiol Renal Physiol. 2007;292:F501–512. doi: 10.1152/ajprenal.00298.2006. PubMed DOI

Di Camillo B, Sanavia T, Martini M, et al. Effect of size and heterogeneity of samples on biomarker discovery: synthetic and real data assessment. PLoS One. 2012;7:e32200. doi: 10.1371/journal.pone.0032200. PubMed DOI PMC

Matheson A, Willcox MD, Flanagan J, Walsh BJ. Urinary biomarkers involved in type 2 diabetes: a review. Diabetes Metab Res Rev. 2010;26:150–171. doi: 10.1002/dmrr.1068. PubMed DOI

Tomita H, Sanford RB, Smithies O, Kakoki M. The kallikrein-kinin system in diabetic nephropathy. Kidney Int. 2012;81:733–744. doi: 10.1038/ki.2011.499. PubMed DOI PMC

Bodin S, Chollet C, Goncalves-Mendes N, et al. Kallikrein protects against microalbuminuria in experimental type I diabetes. Kidney Int. 2009;76:395–403. doi: 10.1038/ki.2009.208. PubMed DOI

Schaffer SW, Jong CJ, Mozaffari M. Role of oxidative stress in diabetes-mediated vascular dysfunction: Unifying hypothesis of diabetes revisited. Vascul Pharmacol. 2012;57(5-6):139–49. PubMed

Zhao L, Gao H, Lian F, et al. (1)H-NMR-based metabonomic analysis of metabolic profiling in diabetic nephropathy rats induced by streptozotocin. Am J Physiol Renal Physiol. 2011;300:F947–956. doi: 10.1152/ajprenal.00551.2010. PubMed DOI

Iori E, Millioni R, Puricelli L, et al. Glycolytic enzyme expression and pyruvate kinase activity in cultured fibroblasts from type 1 diabetic patients with and without nephropathy. Biochim Biophys Acta. 2008;1782:627–633. doi: 10.1016/j.bbadis.2008.08.012. PubMed DOI

Hansen TK, Forsblom C, Saraheimo M, et al. Association between mannose-binding lectin, high-sensitivity C-reactive protein and the progression of diabetic nephropathy in type 1 diabetes. Diabetologia. 2010;53:1517–1524. doi: 10.1007/s00125-010-1742-8. PubMed DOI

Thielens NM, Cseh S, Thiel S, et al. Interaction properties of human mannan-binding lectin (MBL)-associated serine proteases-1 and −2, MBL-associated protein 19, and MBL. J Immunol. 2001;166:5068–5077. doi: 10.4049/jimmunol.166.8.5068. PubMed DOI

Cynis H, Hoffmann T, Friedrich D, et al. The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions. EMBO Mol Med. 2011;3:545–558. doi: 10.1002/emmm.201100158. PubMed DOI PMC

Ruster C, Wolf G. The role of chemokines and chemokine receptors in diabetic nephropathy. Front Biosci. 2008;13:944–955. doi: 10.2741/2734. PubMed DOI

Shirakabe K, Hattori S, Seiki M, et al. VIP36 protein is a target of ectodomain shedding and regulates phagocytosis in macrophage Raw 264.7 cells. J Biol Chem. 2011;286:43154–43163. doi: 10.1074/jbc.M111.275586. PubMed DOI PMC

Huyton T, Gottmann W, Bade-Doding C, et al. The T/NK cell co-stimulatory molecule SECTM1 is an IFN “early response gene” that is negatively regulated by LPS in human monocytic cells. Biochim Biophys Acta. 1810;2011:1294–1301. PubMed

Goldberg S, Harvey SJ, Cunningham J, et al. Glomerular filtration is normal in the absence of both agrin and perlecan-heparan sulfate from the glomerular basement membrane. Nephrol Dial Transplant. 2009;24:2044–2051. doi: 10.1093/ndt/gfn758. PubMed DOI PMC

Morita H, Yoshimura A, Inui K, et al. Heparan sulfate of perlecan is involved in glomerular filtration. J Am Soc Nephrol. 2005;16:1703–1710. doi: 10.1681/ASN.2004050387. PubMed DOI

Mongiat M, Sweeney SM, San Antonio JD, et al. Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan. J Biol Chem. 2003;278:4238–4249. doi: 10.1074/jbc.M210445200. PubMed DOI

Salier JP, Rouet P, Raguenez G, Daveau M. The inter-alpha-inhibitor family: from structure to regulation. Biochem J. 1996;315(Pt 1):1–9. doi: 10.1042/bj3150001. PubMed DOI PMC

Song J, Patel M, Rosenzweig CN, et al. Quantification of fragments of human serum inter-alpha-trypsin inhibitor heavy chain 4 by a surface-enhanced laser desorption/ionization-based immunoassay. Clin Chem. 2006;52:1045–1053. doi: 10.1373/clinchem.2005.065722. PubMed DOI

Lorenzon E, Colladel R, Andreuzzi E, et al. MULTIMERIN2 impairs tumor angiogenesis and growth by interfering with VEGF-A/VEGFR2 pathway. Oncogene. 2012;31:3136–3147. doi: 10.1038/onc.2011.487. PubMed DOI

Nakagawa T, Kosugi T, Haneda M, et al. Abnormal angiogenesis in diabetic nephropathy. Diabetes. 2009;58:1471–1478. doi: 10.2337/db09-0119. PubMed DOI PMC

Klysik J, Theroux SJ, Sedivy JM, et al. Signaling crossroads: the function of Raf kinase inhibitory protein in cancer, the central nervous system and reproduction. Cell Signal. 2008;20:1–9. doi: 10.1016/j.cellsig.2007.07.003. PubMed DOI PMC

Fan Q, Du S, Yang G, et al. Protein expression profile of human renal mesangial cells under high glucose. Am J Nephrol. 2011;34:18–25. doi: 10.1159/000328733. PubMed DOI

Wang H, Feng L, Hu JW, et al. Characterisation of the vitreous proteome in proliferative diabetic retinopathy. Proteome Sci. 2012;10:15. doi: 10.1186/1477-5956-10-15. PubMed DOI PMC

Rocchetti MT, Centra M, Papale M, et al. Urine protein profile of IgA nephropathy patients may predict the response to ACE-inhibitor therapy. Proteomics. 2008;8:206–216. doi: 10.1002/pmic.200700492. PubMed DOI